Unclassified ENV/JM/MONO(99)9

Organisation de Coopération et de Développement Economiques OLIS : 15-Apr-1999Organisation for Economic Co-operation and Development Dist. : 16-Apr-1999__________________________________________________________________________________________

English text onlyENVIRONMENT DIRECTORATEJOINT MEETING OF THE CHEMICALS COMMITTEE AND THE WORKING PARTYON CHEMICALS

Series on Harmonization of Regulatory Oversight in Biotechnology No. 10

CONSENSUS DOCUMENT ON GENERAL INFORMATION CONCERNING THEGENES AND THEIR ENZYMES THAT CONFER TOLERANCE TO GLYPHOSATEHERBICIDE

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Complete document available on OLIS in its original format

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Also published in the Series on Harmonization of Regulatory Oversight in Biotechnology:

No. 1, Commercialisation of Agricultural Products Derived through Modern Biotechnology: SurveyResults (1995)

No. 2, Analysis of Information Elements Used in the Assessment of Certain Products of ModernBiotechnology (1995)

No. 3, Report of the OECD Workshop on the Commercialisation of Agricultural Products Derivedthrough Modern Biotechnology (1995)

No. 4, Industrial Products of Modern Biotechnology Intended for Release to the Environment: TheProceedings of the Fribourg Workshop (1996)

No. 5, Consensus Document on General Information concerning the Biosafety of Crop Plants MadeVirus Resistant through Coat Protein Gene-Mediated Protection (1996)

No. 6, Consensus Document on Information Used in the Assessment of Environmental ApplicationsInvolving Pseudomonas (1997)

No. 7, Consensus Document on the Biology of Brassica Napus L. (Oilseed Rape) (1997)

No. 8, Consensus Document on the Biology of Solanum tuberosum subsp. tuberosum (Potato)(1997)

No. 9, Consensus Document on the Biology of Triticum aestivum (Bread Wheat) (1999)

Consensus Document on General Information Concerning the Genes and their Enzymes which ConferTolerance to Phosphinothricin Herbicides (in preparation)

Consensus Document on the Biology of Picea abies L. (Norway Spruce) (in preparation)

Consensus Document on the Biology of Picea glauca (Moench) Voss (White Spruce)(in preparation)

Consensus Document on the Biology of Populus L. (Poplars) (in preparation)

Consensus Document on the Biology of Oryza sativa (Rice) (in preparation)

Consensus Document on Information Used in the Assessment of Environmental Applications InvolvingRhizobiacea (in preparation)

Consensus Document on Information Used in the Assessment of Environmental Applications InvolvingBacillus (in preparation)

© OECD 1999

Applications for permission to reproduce or translate all or part of this material should be made to:Head of Publications Service, OECD, 2 rue André-Pascal, 75775 Paris Cedex 16, France.

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OECD Environmental Health and Safety Publications

Series on Harmonization of Regulatory Oversight in Biotechnology

No. 10

Consensus Document on General Information Concerningthe Genes and Their Enzymes that Confer Tolerance to

Glyphosate Herbicide

Environment Directorate

Organisation for Economic Co-operation and Development

Paris 1999

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About the OECD

The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental organisation inwhich representatives of 29 industrialised countries in North America, Europe and the Pacific, as well as the EuropeanCommission, meet to co-ordinate and harmonize policies, discuss issues of mutual concern, and work together torespond to international problems. Most of the OECD’s work is carried out by more than 200 specialized Committeesand subsidiary groups composed of Member country delegates. Observers from several countries with special status atthe OECD, and from interested international organisations, attend many of the OECD’s Workshops and othermeetings. Committees and subsidiary groups are served by the OECD Secretariat, located in Paris, France, which isorganised into Directorates and Divisions.

The Environmental Health and Safety Division publishes free-of-charge documents in six different series: Testingand Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides; Risk Management;Harmonization of Regulatory Oversight in Biotechnology; and Chemical Accidents. More information about theEnvironmental Health and Safety Programme and EHS publications is available on the OECD’s World Wide Web site(see below).

This publication is available electronically, at no charge.

For the complete text of this and many other EnvironmentalHealth and Safety publications, consult the OECD’s

World Wide Web site (http://www.oecd.org/ehs/)

or contact:

OECD Environment Directorate,Environmental Health and Safety Division

2 rue André-Pascal 75775 Paris Cedex 16

France

Fax: (33) 01 45 24 16 75

E-mail: ehscont@oecd.org

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FOREWORD

The OECD’s Working1 Group on Harmonization of Regulatory Oversight in Biotechnologydecided at its first session, in June 1995, to focus its work on the development of Consensus Documentsthat are mutually recognised among Member countries. These Consensus Documents contain informationfor use during the regulatory assessment of a particular product. In the area of plant biosafety, ConsensusDocuments are being developed on the biology of certain plant species, on specific genes and resultingproteins that, when introduced into a plant, result in the expression of specific traits, and on biosafetyissues arising from certain general trait modifications made to plants.

This document, which addresses general information concerning the genes and their enzymesthat confer tolerance to the herbicide glyphosate, was prepared by the United States as lead country incollaboration with Germany and the Netherlands. It has been revised based on comments received fromOECD Member countries and on subsequent comments from National Co-ordinators, following a secondround of review in 1998.

The Joint Meeting of the Chemicals Committee and the Working Party on Chemicals hasrecommended that this document be made available to the public. It is published on the authority of theSecretary-General of the OECD.

1. In August 1998, following a decision by OECD Council to rationalise the names of Committees and

Working Groups across the OECD, the “Expert Group on Harmonization of Regulatory Oversight inBiotechnology” became the “Working Group”.

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TABLE OF CONTENTS

Preamble ................................................................................................................................ 9

Summary Note ..................................................................................................................... 11

Section I Herbicide Tolerance ..................................................................................... 13

Section II Glyphosate as a Herbicide ............................................................................ 14

Section III The Development of Glyphosate-Tolerant Plants ........................................ 16

Section IV Genes and Enzymes that Confer Glyphosate Tolerance .............................. 17

Section V Effect of Transgene Expression in Plants..................................................... 19

Section VI References .................................................................................................... 21

Questionnaire to Return to the OECD................................................................................. 25

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Preamble

OECD Member countries are now commercialising and marketing agricultural and industrialproducts of modern biotechnology. They have identified the need for harmonization of regulatoryapproaches for the assessment of these products, in order to avoid unnecessary trade barriers.

In 1993, Commercialisation of Agricultural Products Derived through ModernBiotechnology was instituted as a joint project of the OECD’s Environment Policy Committee and itsCommittee on Agriculture. The objective of this project is to assist countries in their regulatory oversightof agricultural products derived through modern biotechnology – specifically in their efforts to ensuresafety, to make oversight policies more transparent and efficient, and to facilitate trade. The project isfocused on the review of national policies, with respect to regulatory oversight, that will affect themovement of these products into the marketplace.

The first step of this project was to carry out a survey concentrating on national policies inregard to regulatory oversight of these products. Data requirements for products produced through modernbiotechnology, and mechanisms for data assessment, were also surveyed. The results were published inCommercialisation of Agricultural Products Derived through Modern Biotechnology: Survey Results(OECD, 1995).

Subsequently, an OECD Workshop was held in June 1994 in Washington, D.C. with the aim ofimproving awareness and understanding of the various systems of regulatory oversight developed foragricultural products of biotechnology; identifying similarities and differences in various approaches; andidentifying the most appropriate role for the OECD in further work towards harmonization of theseapproaches. Approximately 80 experts in the areas of environmental biosafety, novel food safety andvarietal seed certification, representing 16 OECD countries, eight non-member countries, the EuropeanCommission and several international organisations, participated in the Workshop. Report of the OECDWorkshop on the Commercialisation of Agricultural Products Derived through Modern Biotechnologywas also published by the OECD in 1995.

As a next step towards harmonization, the Working Group on Harmonization of RegulatoryOversight in Biotechnology instituted the development of Consensus Documents that are mutuallyrecognised among Member countries. The purpose of these documents is to describe common elements inthe safety assessment of a new plant variety developed through modern biotechnology, to encourageinformation sharing and prevent duplication of effort among countries. These common elements fall intothree general categories: the biology of the host plant species, or crop; the introduced genes and geneproducts conferring the novel trait; and biosafety issues arising from the introduction of certain generaltrait types into plants.

This Consensus Document is a “snapshot” of current information that may be relevant in aregulatory risk assessment. It is meant to be useful not only to regulatory officials, as a general guide andreference source, but also to industry and others carrying out research and product development.

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It is anticipated that this document and others in the series related to genes and products thatconfer novel traits, together with the relevant Consensus Documents on plant species biology and thoseproviding information on biosafety issues arising from the use of general trait types in plants, will be ofuse in the biosafety assessment of genetically modified plants.

Reference to two other OECD publications that have been published in recent years will alsoprove useful. Traditional Crop Breeding Practices: An Historical Review to Serve as a Baseline forAssessing the Role of Modern Biotechnology presents information concerning 17 different crop plants. Itincludes sections on phytosanitary considerations in the movement of germplasm and on current end usesof the crop plants. There is also a detailed section on current breeding practices. Safety Considerations forBiotechnology: Scale-Up of Crop Plants provides a background on plant breeding, discusses scaledependency effects, and identifies various safety issues related to the release of plants with “novel traits”.1

To ensure that scientific and technical developments are taken into account, OECD countrieshave agreed that Consensus Documents will be updated regularly. Additional areas relevant to the subjectof each Consensus Document will be considered at the time of updating.

Users are therefore invited to provide relevant new scientific and technical information, and tomake proposals concerning additional areas that might be considered in the future. A short, pre-addressedquestionnaire is included at the end of this document. The information requested should be sent to theOECD at one of the addresses shown.

1. For more information on these and other OECD publications, contact the OECD Publications Service, 2 rue

André-Pascal, 75775 Paris Cedex 16, France. Fax: (33) 01.49.10.42.76; E-mail: PUBSINQ@oecd.org; orconsult http://www.oecd.org

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SUMMARY NOTE

This document summarises the information available on the source of the genes that have beenused to construct glyphosate-tolerant transgenic plants, the nature of the enzymes they encode, and theeffects of the enzymes on the plant’s metabolism.

Scope of this document: OECD Member countries agreed to limit this document to a discussionof the introduced genes and resulting enzymes that confer glyphosate tolerance to plants. The document isnot intended to be an encyclopaedic review of all scientific experimentation with glyphosate-tolerantplants. In addition, this document does not discuss the wealth of information available on the herbicideglyphosate itself or the uses of the herbicide in agricultural and other applications. Food safety aspects ofthe use of glyphosate on glyphosate-tolerant transgenic plants are not discussed. Such information isavailable from other sources, including the respective governmental organisations which regulate the useof the herbicide.

While the focus of this document is on the genes and enzymes involved in encoding glyphosatetolerance, reference is not made to specific plant species into which glyphosate tolerance might beintroduced. Any issues relating to the cultivation of glyphosate-tolerant plants or to the potential for, orpotential effects of, gene transfer from a glyphosate-tolerant plant to another crop plant or to a wildrelative are outside the agreed scope of this document. It is intended, however, that this document shouldbe used in conjunction with specific plant species biology Consensus Documents (see list of publicationsat the front of the document) when a biosafety assessment is made of plants with novel glyphosateherbicide resistance.

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Section I - Herbicide Tolerance

Many herbicides kill plants by interfering with enzyme function in the plant. Most of theseherbicides exert their effect on a single enzyme which catalyses a key metabolic reaction in the plant. Ingeneral, plants exhibit a range of sensitivities to the herbicides used in agriculture, with some speciesexhibiting considerable tolerance to a single herbicide. There are several mechanisms by which plants cantolerate exposure to herbicide: (1) the plant produces an enzyme which detoxifies the herbicide; (2) theplant produces an altered target enzyme which is not affected by the herbicide; or (3) the plant producesphysical or physiological barriers to uptake of the herbicide into the plant tissues and cells (Devine et al.1993).

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Section II - Glyphosate as a Herbicide

Glyphosate is widely used as a broad-spectrum weed control agent and is registered in manycountries (Duke 1996, Shah et al. 1986). Even though glyphosate is a reversible competitive inhibitor ofthe enzyme 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSPS) with respect to phosphoenolpyruvicacid (PEP), it does not inhibit any other PEP-dependent enzymatic reactions. It is a non-competitiveinhibitor of EPSPS with respect to 3-phosphoshikimic acid (Steinrucken et al. 1984). Glyphosate isproduced by chemical synthesis. It is not a natural product. Chemically, glyphosate is N-

phosphonomethyl-glycine (see Figure 1). Glyphosate is the active ingredient of the herbicide Roundup®

(Monsanto).

Figure 1 Glyphosate Structure

O O || ||

HO — P — CH2 — NH — CH2 — C — OH

OH

The high sensitivity of crop plants to glyphosate has limited its use as a pre-crop emergenceherbicide in no-till management strategies, and as a herbicide and crop desiccant when applied shortlybefore crop harvest. With the development of genetically engineered crop plants that are resistant toglyphosate, this herbicide can instead be applied after both crops and weeds have emerged, with little orno damage to the crop.

Glyphosate interferes with normal plant metabolism through inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSPS). In plants and micro-organisms, EPSPS isinvolved in the biosynthesis of aromatic amino acids, vitamins, and many secondary metabolites. It is notpresent in animals (Levin and Sprinson 1964, Steinrucken and Amrhein 1980). In plants, EPSPS islocalised within plastids. This enzyme condenses phosphoenolpyruvic acid (PEP) and 3-phosphoshikimicacid to 5-enolpyruvyl-3-phosphoshikimic acid. As a consequence of the inhibition of aromatic amino acidbiosynthesis, protein synthesis is disrupted, resulting in the plant’s death (Kishore and Shah 1988). Whilesome of the downstream products of the EPSPS reaction, amino acids and vitamins, are strictly essentialfor the growth of all living organisms, some secondary metabolites derived from the shikimate pathwaymay have specific survival value for the producing organism (Malik 1986). The enzyme has rigidspecificity towards its substrates, which are shikimate-3-phosphate and phosphoenolpyruvate (Andersonand Johnson 1990). The reaction product, 5-enolpyruvylshikimate-3-phosphate (EPSP), is further actedupon by other enzymes to yield chorismic acid, which gives anthranilic acid (a precursor of tryptophan)and, on rearrangement, prephenic acid (a precursor of phenylalanine and tyrosine).

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Based on the knowledge of the mode of action of glyphosate, several strategies have emerged fordeveloping plants that are tolerant of exposure to the herbicide. The two successful strategies to produceglyphosate-tolerant plants are introduction of glyphosate-tolerant EPSPS and introduction of an enzymethat inactivates glyphosate, glyphosate oxidoreductase (GOX). Recombinant DNA techniques have beenused to express genes that encode glyphosate-tolerant EPSPS enzyme alone or a combination of EPSPSand GOX genes in susceptible plants (Nida et al. 1996, Padgette et al. 1995, 1996).

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Section III - The Development of Glyphosate-Tolerant Plants

Scientists have been unsuccessful in producing glyphosate-tolerant plants using classicaltechniques. Traditional mutagenesis and selection techniques have to date failed to produce a useful levelof tolerance in crop plant species, although such an approach could yield a mutant form of the targetenzyme that is tolerant of the herbicide but retains its desirable enzymatic function. Plant breeders alsohave been unable to develop glyphosate-tolerant crops using the standard techniques in which chemical orradiation exposure of seeds generates mutations in the plant genome. In cases where the desiredphenotype is herbicide tolerance, spraying seedlings in the growth chamber or field can sometimes beused with success to select tolerant individual plants from millions of mutagenized individuals. Eventhough this approach has been used in the commercial development of imidazolinone-tolerant maize andsoybean cultivars, it has not been successful in producing glyphosate-tolerant plants. This is because allmutant EPSPS, in parallel to glyphosate tolerance, has decreased affinity for phosphoenolpyruvate. Thishas resulted in glyphosate-tolerant plants that have invariably shown reduced biosynthesis of aromaticamino acids.

Recombinant DNA techniques have been used to confer glyphosate tolerance to a variety of cropplant species. In this approach, plants have been transformed with genes that encode a glyphosate-tolerantenzyme that is not inhibited by glyphosate but provides substrates for the biosynthesis of amino acids. Insome cases, the tolerance imparted by this gene has been further augmented by expressing a second genethat encodes the enzyme glyphosate oxidoreductase (GOX) to detoxify glyphosate (Padgette et al. 1996,Shah et al. 1986).

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Section IV - Genes and Enzymes that Confer Glyphosate Tolerance

Three genes which provide field-level tolerance to glyphosate, the active ingredient in Roundup®

herbicide, have been introduced into commercial cultivars. The first glyphosate-tolerant EPSPS gene wasisolated from a soil bacterium, Agrobacterium (Barry et al. 1994, Duke 1996). The EPSPS synthase fromthis Agrobacterium was highly tolerant to glyphosate. When it is expressed in transgenic plants, theEPSPS encoded by this Agrobacterium gene fulfills the aromatic amino acid needs of the plant in thepresence of glyphosate, whereas the plant version of this enzyme (ubiquitous in nature) is sensitive toglyphosate. Agrobacterium spp. are not human or animal pathogens, but some species are pathogenic toplants (Croon 1996, Holt 1984).

Recently, the EPSPS gene from corn (Zea mays) has been mutagenized in vitro to obtain aglyphosate-tolerant enzyme. The tolerant version of the enzyme produced by the modified maize gene is99.3% identical to the parent enzyme (Monsanto 1997).

Also, a gene that encodes for a glyphosate-degrading enzyme called glyphosate oxidoreductase(GOX) was isolated from Achromobacter strain LBAA, a soil bacterium ubiquitous in nature (Barry et al.1994). The encoded enzyme deactivates the herbicidal effect of glyphosate. Glyphosate oxidoreductasecatalyses the conversion of glyphosate to aminomethylphosphonic acid (AMPA) and glyoxylate. GOXrequires flavin adenine dinucleotide (FAD) and magnesium for activity; therefore, it is more appropriatelydesignated an apoenzyme.

EPSPS enzyme, the target of glyphosate action, is synthesised in the cytoplasm and thentransported to the chloroplast (Kishore and Shah 1988). The translocation of the protein to the chloroplastis carried out by an N-terminal protein sequence called the chloroplast transit peptide (CTP). CTPs aretypically cleaved from a mature protein and degraded following delivery to the plastid (Della-Cioppa et al.1986). A plant-derived coding sequence expressing a chloroplast transit peptide is often linked with eachof the genes imparting glyphosate tolerance. This peptide facilitates the import of the newly translatedenzymes into the chloroplasts, the site of both the shikimate pathway and glyphosate mode of action.

Use of the technology achieving transgene expression in plants is now routine. In order toachieve efficient expression of bacterial genes within plants, it has been common for researchers tomodify the codon usage pattern of genes of bacterial origin prior to introducing them into plants. In thiscase, the codon usage pattern of the Agrobacterium glyphosate-tolerant EPSPS gene and glyphosateoxidase genes of Achromobacter have been chemically synthesised for codon optimisation for efficientexpression in the plant. The amino acid sequence of the resulting enzymes is not changed. The genesassociated with their transit peptide coding sequence are usually linked to other regulatory sequences likepromoters, terminators, enhancers and introns. These regulatory sequences do not usually encode for aprotein (Croon 1996).

These genes have been engineered (singly or in combination) into many plant species for thedevelopment of glyphosate tolerance and for use as selectable markers for identification of transformedplants. Plants field-tested with these genes include: Beta vulgaris (beet), Zea mays (corn), Gossypium

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hirsutum (cotton), Lactuca sativa (lettuce), Populus (poplar), Solanum tuberosum (potato), Brassica napus(oilseed rape, rapeseed, canola), Glycine max (soybean), Nicotiana tabacum (tobacco), Lycopersiconesculentum (tomato), Triticum aestivum (wheat).

OECD Member countries have governmental organisations which regulate the field-testing andunrestricted release of genetically engineered plants. Information about these plants is shared amongvarious Member countries. The OECD sponsors an electronic database format for the exchange of thisinformation. The database information is periodically updated to provide information that is both currentand accurate (www.oecd.org/ehs/service.htm).

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Section V - Effect of Transgene Expression in Plants

During the life cycle of any herbicide-tolerant plant, the plant is exposed only rarely to theherbicide. Except for the production of the enzyme(s) encoding glyphosate tolerance, there should be noother changes in plant metabolism. After glyphosate application, the enzyme activities expressed by thetransgenes enable the plant to survive herbicide exposure. In the case of introduced EPSPS, no newmetabolic products are formed since the only difference from the native enzyme is its insensitivity toglyphosate. However, if very high expression levels result from the insertion, the levels of downstreammetabolites might change. In contrast, GOX will convert glyphosate to aminomethylphosphonic acid(AMPA) and glyoxylate when glyphosate herbicide is applied (Torstensson 1985). Since glyoxylate is anaturally occurring plant metabolite involved in carbon cycling, it will be further metabolised to provideintermediates for the Krebs cycle. Since GOX is highly specific for its substrate, glyphosate, in theabsence of glyphosate no metabolites are expected. The United States Environmental Protection Agencyhas decided that only glyphosate residues are to be regulated in plant and animal commodities, and thatthe major metabolite AMPA is not of toxicological concern regardless of its level in food (US EPA 1997).Information regarding decisions concerning glyphosate herbicide-tolerant plants can be found at:

http://www.olis.oecd.org/bioprod.nsfhttp://www.cfia-acia.agr.ca/english/plant/pbo/home_e.html (Canada)http://ss.s.affrc.go.jp/docs/sentan/eguide/commerc.htm (Japan)http://www.aphis.usda.gov/biotech/petday.html (USA)http://europa.eu.int/comm/dg24/health/sc/scp/outcome_en.html (European Commission)

Western blot and enzymatic activity assays indicate that EPSPS protein from Agrobacteriumstrain CP4 is readily degraded in less than two minutes by incubation in simulated gastric fluid. Insimulated intestinal fluid the enzyme activity and immunoreactivity lasts longer, being still detectable atten minutes but undetectable at 270 minutes. The GOX protein is rapidly degraded in simulated gastricfluid and simulated intestinal fluid. After a 15 second incubation in gastric fluid, GOX has less than 90%of its initial protein epitopes as assayed by Western blot analysis, and enzyme activity loss is also greaterthan 90% when assayed after one minute incubation in gastric fluid. Similar results are seen in simulatedintestinal fluid (US EPA 1996 and 1997).

Expression of GOX and glyphosate-tolerant EPSPS is not detrimental to plant growth, sincesuch crops have agronomic performance similar to their parents. Governmental regulatory agencies in theUnited States (US Department of Agriculture 1994, 1995, 1997), Canada (Agriculture and AgrifoodCanada 1995, 1996), Japan (Ministry of Agriculture, Forestry and Fisheries 1996) and European Union(European Commission 1998a, b) have made decisions that the presence of the EPSPS and GOX proteinsin plants does not result in plants that are unsafe in their environments. Several lines of evidence supportthe conclusion that these enzymes show low mammalian toxicity: (1) Neither enzyme shows amino acidhomology to known allergens or mammalian toxins (Burke and Fuchs 1996); (2) Data from acute oraltoxicity tests at high concentration of enzymes showed no toxicity (Harrison et al. 1996). In acute oraltoxicity tests of bacterially derived CP4 EPSPS protein, no test substance adverse effects occurred at adose of 572 milligrams per kilogram body weight (mg/kg) of the test animals. The acute toxicity of

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bacterially derived GOX protein showed no test substance adverse effects at doses of 91.3 mg/kg of thetest animals; (3) Both enzymes are readily inactivated by heat or mild acidic conditions and are readilydegraded in an in vitro digestibility assay which is consistent with the lack of oral toxicity (US EPA 1996,1997). That the two enzymes show little if any toxicity is consistent with the observation that mostenzymes are not considered toxic to vertebrates (Kessler et al. 1992). Notable exceptions are diphtheriatoxin and certain enzymes in the venom of snakes, with very different exposure scenarios.

Governmental regulatory agencies in the United States (US Food and Drug Administration1996), Canada (Agriculture and Agrifood Canada 1995, 1996), Japan and the European Union have madedecisions that the presence of the EPSPS and GOX proteins in plants released into the environment do notpose a significant allergenicity risk. Two independent lines of evidence support the decision that theseenzymes are not potential allergens: (1) Current scientific knowledge suggests that common foodallergens tend to be resistant to degradation by heat, acid and proteases, are glycosylated, and are presentat high concentrations in food. The EPSPS and GOX proteins are rapidly degraded by gastric fluid in vitroand are non-glycosylated. Thus, the potential for these proteins to be food allergens is minimal (Astwoodet al. 1996, Burke and Fuchs 1996); (2) It is possible to utilise international gene databases to compare thegene sequences of a protein with other genes that encode known allergens. None of the amino acidsequences of known allergens or proteins involved in disease were shown to have similarity to the EPSPSor GOX proteins, as defined by eight identical and contiguous amino acids in a sequence. Likewise, noneof the amino acid sequences of known allergens or proteins involved in coleiac disease were shown tohave similarity to the GOX protein as defined by eight contiguous amino acids in a sequence (US EPA1997).

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Section VI - References

Agriculture and Agri-food Canada (as of 1 April 1997, the Canadian Food Inspection Agency). 1995.Monsanto Canada Inc.’s Glyphosate-tolerant soybean (Glycine max L.) Line GTS 40-3-2. DD95-05.

(Available electronically at http://www.cfia-acia.ca/english/food/pbo/bhome.html)

Agriculture and Agri-food Canada, Food Production and Inspection Branch (as of 1 April 1997, theCanadian Food Inspection Agency). 1996. Monsanto Canada Inc.’s Roundup Herbicide-Tolerant Brassicanapus Canola line GT200. DD96-07.

(Available electronically at http://www.cfia-acia.ca/english/food/pbo/bhome.html)

Anderson, K.S. and Johnson, K.A. 1990. Kinetic and structural analysis of enzyme intermediates: lessonsfrom EPSP synthase. Chem. Rev. 90: 1131-1149.

Astwood, J.D., Leach, J.N. and Fuchs, R.L. 1996. Stability of food allergens to digestion in vitro. NatureBiotechnology 14:1269-1273.

Barry, G., Kishore, G., Padgette, S., Taylor, M., Kolacz, K., Weldon, M., Re, D., Eichholtz, D.,Fincher, K. and Hallas, L. 1992. In: Singh, B.K., Flores, H.E. and Shannon, J.C. (eds.), Biosynthesis andMolecular Regulation of Amino Acids in Plants. American Society of Plant Physiologists, Madison,Wisconsin, pp. 139-145.

Barry, G.F., Taylor, M.L., Padgette, S.R., Kolacz, K.H., Hallas, L.E., Della-Cioppa, G. and Kishore, G.M.1994. Cloning and Expression in Escherichia coli of the Glyphosate-to-Aminomethylphosphonic AcidDegrading Activity from Achromobacter sp. strain LBAA. Monsanto Technical Report MSLB13245,St. Louis.

Burke, A.W. and Fuchs, R.L. 1996. Assessment of the endogenous allergens in glyphosate-tolerant andcommercial soybean varieties. J. Allergy and Clinical Immunology 96:1008-1010.

Croon, K.S. 1996. Petition for Determination of Nonregulated Status: Insect-protected Roundup ReadyCorn Line MON 802. USDA Petition No. 96 - 317 - 01P.

Della-Cioppa, G., Bauer, S.C., Klein, B.K., Shah, D.M., Fraley, R.T. and Kishore, G. 1986. Translocationof the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants invitro. Proc. Natl. Acad. Sci. U.S.A. 83: 6873-6877.

Della-Cioppa, G., Bauer, S.C., Taylor, M.T., Rochester, D.E., Klein, B.K., Shah, D.M., Fraley, R.T. andKishore, G.M. 1987. Targeting a herbicide-resistant enzyme from Escherichia coli to chloroplasts ofhigher plants. Bio/Technology 5: 579-584.

Devine, M., Duke, S.O. and Fedtke, C. 1993. Physiology of Herbicide Action. Prentice Hall, EnglewoodCliffs, NJ, pp. 251-294.

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Duke, S.O. 1996. Herbicide resistant crops. CRC Press, New York.

European Commission, Directorate General XXIV, Policy and Consumer Health Protection: ScientificCommittee on Plants. 1998a. Opinion of the Scientific Committee on Plants regarding submission forplacing on the market of fodder beet tolerant to glyphosate notified by DLF-Trifolium, Monsanto andDanisco Seed (notification C/K/97/01)(Opinion expressed by SCP on 23 June 1998).

(See: http://europa.eu.int/comm/dg24/health/sc/scp/out16_en.htm)

European Commission, Directorate General XXIV, Policy and Consumer Health Protection: ScientificCommittee on Plants. 1998b. Opinion of the Scientific Committee on Plants regarding the geneticallymodified cotton tolerant to glypohosate herbicide notified by the Monsanto Company (notificationC/ES/97/01) (Opinion expressed by the SCP on 14 July 1998).

(See: http://europa.eu.int/comm/dg24/health/sc/scp/out17_en.htm)

Hallas, L.E., Hahn, E.M. and Korndorfer, C. 1988. Characterization of microbial traits associated withglyphosate biodegradation in industrial activated sludge. J. Ind. Microbiol. 3: 377-385.

Harrison, L.A., Bailey, M.R., Naylor, M.W., Ream, J.E., Hammond, B.G., Nida, D.L., Burnette, B.L.,Nickson, T.E. and Mitsky, T.A. 1996. Recombinant 5-enolpyruvylshikimate 3-phosphate synthase inglyphosate-tolerant soybeans is digestible. J. Nutr. 126 (3) 728-40.

Holt, J.G., editor-in-chief. 1984. Bergy’s Manual of Systematic Bacteriology. William and Wilkins,Baltimore.

Japan, Ministry of Agriculture, Forestry and Fisheries. 1996. Current Status of Commercializationof Transgenic Crop Plants in Japan.

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QUESTIONNAIRE TO RETURN TO THE OECD

This is one of a series of OECD Consensus Documents that contain information for use during theregulatory assessment of particular micro-organisms, or plants, developed through modernbiotechnology. Consensus Documents are developed with the intention that they will be updatedregularly, in order to reflect scientific and technical developments.

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